The main goal of the proposed research is to understand the dynamics of transcriptional activation of human immunodeficiency virus type-1 (HIV-1) gene expression. HIV-1 encodes a transcriptional transactivator protein called Tat, which is expressed early in the viral life cycle and is absolutely required for viral replication and progression to disease. A regulatory element between +1 and +60 in the HIV-1 long terminal repeat which is capable of forming a stable stem-loop structure designated TAR is critical for Tat function. Tat-TAR interactions convert RNA polymerase II (RNA pol II) enzyme complex into its processive form and lead to the efficient production of full-length viral transcripts. How is the RNA pol II processivity regulated? What is the role of RNA-protein interactions in transactivation? What is the relationship between the dynamics of RNA structure and transcription attenuation? What other cellular proteins interact with the transcription elongation complex and TAR RNA and how do they control the processivity of RNA pol II? When do the cellular proteins join and leave the elongating RNA pol II complex? The proposed research aims to address these questions, since answers to these questions would greatly improve our understanding of the biology of HIV 1. A clear understanding of the novel mechanisms which control transcription elongation of HIV-1 and other genes is fundamental to understanding normal development as well as such diseases as cancer and AIDS. Our project has three specific aims. Specific aim 1: Isolation of elongation complexes. Experiments are proposed to develop general methods for isolating a homogeneous population of RNA polymerase II elongation complexes arrested at a DNA damage site. DNA damage will be introduced by a psoralen crosslink in DNA templates containing HIV-1 promoter. After performing transcription, elongation complexes will be purified and characterized. Specific Aim 2: RNA-protein interactions during transcription. Experiments are proposed to introduce modified nucleotides into the RNA sequences and perform photocrosslinking studies to isolate cellular proteins that interact with RNA. Specific Aim 3: Protein-protein interactions during transcription. Experiments are proposed to site-specifically modify protein side chains with photoactive groups and perform photocrosslinking studies to visualize protein-protein interactions.